Certain cells of the immune system have cytotoxic activity against particular target cells. Cytotoxic T lymphocytes (CTLs) are specifically directed to their targets via antigen-derived peptides bound to MHC class I-specific markers. Natural killer (NK) cells, however, are not so restricted. NK cells, generally representing about 10-15% of circulating lymphocytes, bind and kill target cells, including virus-infected cells and many malignant cells, nonspecifically with regard to antigen and without prior immune sensitization (Herberman et al., Science 214.24 (1981)). Killing of target cells occurs by inducing cell lysis. MHC class restriction likewise is not involved. In these ways the activity of NK cells differs from antigen-specific and MHC class-specific T cells, such as cytotoxic T lymphocytes. Use of NK cells in the immunotherapy of tumors and malignancies is suggested by these properties, since many tumors are MHC class I deficient and therefore do not attract CTL activity. Adhesion molecules may also be involved in the targeting of NK cells; for example, it is observed that the Fey receptor (CD16) is expressed on NK cells. NK cells are large granular lymphocytes which lack CD3, and in addition to CDI6, also may express Leu19 (Lanier et al., J. Immunol. 136; 4480 (1986)).
NK cells are activated when exposed to cytokines such as interleukin-2 (IL-2), IL-7, IL-12, and interferons (Alderson et al., J. Exp. Med. 172:577-587 (1990); Robertson et al., J. Exp. Med. 175:779-788 (1992)). The resulting cells are called lymphokine activated killer (LAK) cells. The spectrum of target cells is altered in activated NK cells compared to nonactivated cells, although the mechanism of killing may be identical or similar (Philips et al., J. Exp. Med. 164:814-825 (1986)).
More generally, killing activity in the cells of the immune system may be induced by treating a population of cells, such as peripheral blood mononuclear cells (PBMCs), with lymphokines. Such preparations contain LAK cells. LAK cells may also be generated from autologous samples of peripheral blood lymphocytes. LAK cells have antitumor killing activity while having essentially no effect on normal cells. They appear to purge leukemia (Long et al., Transplantation 46:433 (1988); Xhou et al., Proc. Am. Assoc. Cancer Res. 34:469 (1993; abstract)), lymphoma (Schmidt-Wolf et al., J. Exp. Med. 174: 139 (1991); Gambacorti-Passerini et al., Br. J. Haematol. 18:197 (1991)) and neuroblastoma (Ades et al., Clin. Immunol. Immunopathol. 46:150 (1988)). NK cells, activated NK cells, and LAK cells are distinguishable by their cell surface markers and by the identity of the target cells that they kill.
Activated and expanded (i.e., cultured to proliferate) NK cells and LAK cells have been used in both ex vivo therapy and in vivo treatment in patients with advanced cancer. Some success with ex vivo therapy has been observed in bone marrow related diseases, such as leukemia, breast cancer and certain types of lymphoma. In vivo treatment may be directed toward these and other forms of cancer, including malignant melanoma and kidney cancer (Rosenberg et al., N. Engl. J. Med. 316:889-897 (1987)). LAK cell treatment requires that the patient first receive IL-2, followed by leukophoresis and then an ex vivo incubation and culture of the harvested autologous blood cells in the presence of IL-2 for a few days. The LAK cells must be reinfused along with relatively high doses of IL-2 to complete the therapy. This purging treatment is expensive and can cause serious side effects. These include fluid retention, pulmonary edema, drop in blood pressure, and high fever. In some cases in which these side effects occur, intensive care unit management is required.
Purging techniques have been applied in other circumstances as well. Cytotoxic drugs or monoclonal antibodies combined with complement, and toxins, may be administered in order to bring about remission. In such cases bone marrow or blood stem cells, purged to reduce the number of residual leukemic cells present, have been infused back into the patient after the drug treatment (Uckun et al., Blood 79:1094 (1992)). Gene marking studies have shown that unpurged bone marrow may contribute to relapse in patients presumed to be in remission (Brenner et al., Lancet 341:85 (1993)). This suggests that some form of purging of autologous marrow or blood prior to transplantation is necessary (Klingemann et al., Biol. Blood Marrow Transplant. 2:68-69 (1996)).
Recently, preclinical studies have also demonstrated promising antitumor activity in vivo with a lethally irradiated, MHC-unrestricted, cytotoxic T-cell leukemic clone (TALL-104) (Cesano et al., Cancer Immunol. Immunofher. 40:139-151 (1995); Cesano et al., Blood 87:393-403 (1996)). These cells were derived from leukemia T cell lines obtained from patients having acute T lymphoblastic leukemias (ALL). They bear the CD3 cell surface marker, but not the CD56 marker, in distinction to NK cells which have the converse immunophenotype (CD3− CD56+). Adoptive transfer of these cells was able to eliminate human leukemic cell lines in xenografted severe combined immunodeficient (SCID) mice and to induce remissions of spontaneous lymphomas in dogs without producing T-cell leukemia in the animal models (Cesano et al. (1995); Cesano et al. (1996); Cesano et al., J. Clin. Invest. 94:1076-1084 (1994); Cesano et al., Cancer Res. 56:3021-3029 (1996)).
In spite of the advantageous properties of NK cells in killing tumor cells and virus-infected cells, they remain difficult to work with and to apply in immunotherapy. It is difficult to expand NK cells ex vivo that maintain their tumor-targeting, tumoricidal, and viricidal capabilities in vivo. This remains a major obstacle to their clinical use in adoptive cell immunotherapy (Melder et al., Cancer Research 48:3461-3469 (1988); Stephen et al., Leuk. Lymphoma 377-399 (1992); Rosenberg et al., New Engl. J. Med. 316:889-897 (1987)). Studies of the mechanisms whereby NK cells exert their tumoricidal and viricidal effects are also limited by difficulties in enriching the NK cell fractions without compromising their biological functions and in obtaining pure NK cells that are not contaminated by T cells or other immune effector cells. In an attempt to overcome these problems, many investigators have turned to the use of established NK-like cell lines to explore the mechanisms whereby target cells are susceptible to cytotoxic cells (Hercend et al., Nature 301:158-160 (1983); Yodoi et al., J. Immunol. 134:1623-1630 (1985); Fernandez et al., Blood 67:925-930 (1986); Robertson et al., Exp. Hematol. 24:406-415 (1996); Gong et al., Leukemia 8:652-658 (1994)). NK cell lines described in earlier work carry T lymphocyte-associated surface markers, in spite of the fact that they were developed from precursor cells depleted of T cells (Rosenberg, et al. (1987); Hercend, et al., (1983)).
There thus remains a need for a method of treating a pathology related to cancer or a viral infection with a natural killer cell line that maintains viability and therapeutic effectiveness against a variety of tumor classes. This need encompasses therapeutic methods in which samples from a mammal are treated ex vivo with natural killer cells, as well as methods of treatment of these pathologies with natural killer cells in vivo in a mammal. There is also a need for a natural killer cell line that maintains its own propensity for viability and cytolytic activity by secreting a cytokine which fosters these properties. There also remains a need for such natural killer cell lines which are modified to be more effective, convenient, and/or useful in treatment of cancer and viral infection. It is the objective of this invention to provide NK cells and methods that address these needs.